![Figure 2. Plk1 and Cdk1 regulate MISP phosphorylation. (A) GST-MISP or GST was incubated with His-Plk1 and γ-[32P]ATP (±100 nM of the Plk1 inhibitor BI2536) and analyzed by autoradiography. Coomassie staining shows equal loading. P-GST-MISP, phosphorylated GST-MISP. (B) HeLa cells were transfected with Plk1 or control siRNA for 36 h followed by 16 h of nocodazole treatment. Western blot analysis was performed with indicated antibodies. Cyclin B, loading control. (C) GST-MISP wild type (WT), 7AP mutant, or GST was incubated with His-Plk1 in the presence of γ-[32P]ATP followed by autoradiography. Equal loading is shown by Coomassie staining. (D) GST-MISP WT, 9AC mutant, or GST was incubated with GST-cyclin B/His-Cdk1 in the presence of γ-[32P]ATP followed by autoradiography. Equal loading is shown by Coomassie staining. (E) Flag-MISP WT/7AP/9AC or Flag alone was overexpressed and immunoprecipitated from mitotic HeLa cells. Far Western blot was performed using GST-Plk1 PBD and analyzed with indicated antibodies.](https://cdn.rupress.org/rup/content_public/journal/jcb/200/6/10.1083_jcb.201207050/5/jcb_201207050_fig2.jpeg?Expires=1771735483&Signature=jr1Qjaz5rN08Opxwql23RmcRzb3xFLYkIKO2tCgP~qgmN36XRBlcmRre5u55pLIvNmmtmHZiNU40a~21ArKKa27Zi85GVCjjAg6QYfa82Lx48Lh0BbuZNVC20O3rTcaF~hlrw50RqeV6xBtq~L4nP4ueqVsVlOrcmjJIaOZYxtLFoPGNGkMvEuwPjArprhqPRevPEN2OayJ6yQ1tveRRsIgqmX9cAeEknUmurzyDzfIl5TSfLiUzcB6vQBVOSW4xzDM8op8NezKiY5W63r4cToggUStBrmHOFlloPlLHzICE3FFhpYJ7Up8WEj2t0APj3GfJkNgLj1bXHGCqJ7n1Iw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Plk1 and Cdk1 regulate MISP phosphorylation. (A) GST-MISP or GST was incubated with His-Plk1 and γ-[32P]ATP (±100 nM of the Plk1 inhibitor BI2536) and analyzed by autoradiography. Coomassie staining shows equal loading. P-GST-MISP, phosphorylated GST-MISP. (B) HeLa cells were transfected with Plk1 or control siRNA for 36 h followed by 16 h of nocodazole treatment. Western blot analysis was performed with indicated antibodies. Cyclin B, loading control. (C) GST-MISP wild type (WT), 7AP mutant, or GST was incubated with His-Plk1 in the presence of γ-[32P]ATP followed by autoradiography. Equal loading is shown by Coomassie staining. (D) GST-MISP WT, 9AC mutant, or GST was incubated with GST-cyclin B/His-Cdk1 in the presence of γ-[32P]ATP followed by autoradiography. Equal loading is shown by Coomassie staining. (E) Flag-MISP WT/7AP/9AC or Flag alone was overexpressed and immunoprecipitated from mitotic HeLa cells. Far Western blot was performed using GST-Plk1 PBD and analyzed with indicated antibodies.
